Project Summary/Abstract Genetic reprogramming of cell fate and identity is a promising strategy for cell-based therapies and tissue regeneration. In particular, targeted manipulation of a few genes has been shown as a useful method for induced pluripotent stem (iPS) cell derivation, stem cell differentiation, and T-cell reengineering. Though rapid progress has been made, current methods for targeted genome manipulation are very inefficient and require many accessory factors, limiting their utility for effective cell reprogramming to control different levels of regulation and to coordinate the kinetic expression of large numbers of genes. The major goal of the research is to develop a novel modular and programmable RNA-guided platform that can be used to target multiple genes in a genome for transcriptional or epigenetic regulation, and to exploit its applications for iPS cell reprogramming and potentially other cell-based therapeutic approaches. The proposed platform is built on my recently demonstrated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system derived from the bacterial immune pathway, which is an RNA-guided method for transcriptional silencing of arbitrary genes in diverse host cells. The method requires only a single modified Cas protein (catalytically inactive) and a designed small guide RNA with a 20-basepair complementary region to any gene of interest, without genetically altering the target locus. To further develop the system as a novel cell-reprogramming platform, I will first perform high-throughput characterization experiments in bacteria as a model to quantify the determinants of RNA-guided regulatory efficiency and specificity. Mathematical models will be developed and used for the rational design of large RNA libraries for efficient and specific genome targeting. Second, to develop a programmable platform for various regulatory functions, I will extend the CRISPRi system as a modular DNA-binding system for use in mammalian cells. The system will be combined with protein effectors for different types of genome-scale targeted regulations, including transcription activation, transcription silencing, and heritable histone modification. I will also introduce the ability to regulate these functions using light, through the use of optogenetically controlled protein interactions. Third, as a test bed, I will focus on using the programmable CRISPRi platform as an alternative and perhaps easier way to generate iPS cells. I will create light-gated transcription circuits to precisely control the expression program of endogenous transcription factors that are known to be important for iPS cell reprogramming. I will also use the CRISPRi platform to target and modify epigenetic regulation of these factors, and study if regulating epigenetic marks could achieve more efficient, more stable, and safer reprogramming. Together, these aims will address a critical barrier for cell reprogramming by providing a novel RNA-guided platform for sequence-specific regulation of multiple genes for various types of regulation. Further, the proposal will provide a novel technological basis for constructing gene circuits to coordinate multiple genes for iPS cell generation, which is also directly applicable to other cell reprogramming applications such as stem cell differentiation and T-cell engineering.